1. Field of the Invention
This disclosure is concerned generally with methods of detecting and/or determining the concentration of various substances found in fluids, especially human body fluids. More specifically, the disclosure is concerned with particulate carriers used in methods of determining such substances via solid-phase immunoassay techniques.
2. Prior Art
The expression "immunoassay", as used herein, refers to a method of determining the presence or concentration of a substance in a fluid, which method is based on the use of antibodies specific to that substance. Since it is known that antibodies to a given substance are extremely specific to that substance, research efforts have been directed in recent years to use that specificity in determining the presence or concentration of substances which are present in very small quantities in fluids, especially human body fluids such as blood. Although there now exists a wide variety of immunoassay techniques, the more common assays require the use of a label for either the antibody or the antigenic substance or hapten being determined. The use of a label permits a relatively rapid detection or quantitation in conventional laboratories using conventional equipment. A variety of labels is known and used in immunoassays. For example, fluorogenic materials useful in a fluoroimmunoassay (FIA) are described in U.S. Pat. No. 3,940,475 to Gross. Enzyme markers can be coupled to antibodies or antigens to perform an enzyme immunoassay (EIA) as illustrated in U.S. Pat. No. 3,654,090 to Schuurs et al. Radioisotopes can be incorporated into an antibody or substance to perform a radioimmunoassay (RIA) as illustrated in U.S. Pat. No. 3,555,143 to Axen et al. As used herein, the expression "labeled antibody" or its equivalent includes any of those known labels.
A typical immunoassay requires, at some point, an immunochemical complexation between an antigenic substance and its respective antibody. Commonly, one of the species in such a complexation is labeled, and, by competing with, complexing with, or displacing an unknown substance in such complexation, and then quantitating the label (e.g., fluorometrically, enzymatically, radiometrically, etc.), it is possible to determine the unknown by known means. Prior to such quantitation, however, it is generally necessary to separate the immunochemically complexed products from the surrounding incubation medium. Such separations can be facilitated by providing one of the species involved in an immobilized, insoluble form. For example, it is known that antigenic substances, haptens or antibodies thereto can be attached to, or incorporated in, various water-insoluble carrier materials without substantial loss of biological activity. See, for example, U.S. Pat. Nos. 3,555,143 (organic carriers) and 3,652,761 (inorganic carriers). When either of the reactants in an immunoassay is used in such an immobilized form, there is present a solid phase which, when appropriate, can be readily separated (e.g., by centrifugation or filtration) for label quantitation. The use of composites comprising antibodies or antigens associated with or immobilized on essentially water-insoluble carrier materials is commonly referred to as a solid-phase immunoassay (SPIA). As used herein, the expression "immobilized antibody composite" or the equivalent includes all forms of antibodies which have been attached to insoluble materials.
Porous glass is used as the water-insoluble carrier phase in a number of SPIA systems available commercially from Corning Medical, Corning Glass Works, Medfield, Mass.; for example, T-4 (thyroxine) and TSH (thyrotropin) RIA test systems.
The type of porous glass used in the above-identified RIA's is a "controlled-pore glass" formed by leaching a borosilicate glass. See "Controlled-Pore Glasses for Enzyme Immobilization", by Filbert, as Chapter 3 in Immobilized Enzymes for Industrial Reactors, Messing (ed.), Academic Press (1975), describing preparation, composition, physical, chemical, and mechanical properties and surface chemistry, including covalent bonding of biologicals to the controlled-pore glass, of such glasses. Although non-porous glass could be employed as a carrier phase, the porous glass offers obvious advantages in providing a high surface area per unit volume.
Solid phase carrier materials are normally used in finely-divided particulate form for two main reasons. Firstly, in order to make assays quantitative, several known concentrations of the species being assayed are measured, along with the samples to be assayed, forming a batch of test samples. By carefully ensuring that all test samples are treated identically, the known concentrations provide a standard or calibration curve for that particular batch. By attaching the reagent to finely-divided particles, which are dispersed in liquid in a gently stirred receptacle, each predetermined volume drawn from the receptacle contains the same quantity of reagent. Secondly, most assays involve an incubation period during which the immobilized reagents react with other reagents in solution. It is desirable that the immobilized reagent remains dispersed in suspension without serious sedimentation so that dissolved reagents do not have to diffuse very far to reach immobilized material. Conversely, the difficulty of centrifuging down very fine particles leads to "fines" being thrown away as undesirable. From these considerations the present practice of using porous glass particles of perhaps 0.5 to 3 microns diameter and 50% to 70% by volume porosity has emerged.
As noted hereinbefore, centrifugation is routinely used to separate the solid phase from the reaction fluid. The fluid is manually or mechanically decanted. Depending upon the centrifuge available, number of samples being assayed, and so on, separation can take up to 10 minutes or more.
Magnetic separation has been considered in the art to avoid the need for centrifugation. In this type of procedure, the water-insoluble solid carrier is a magnetic particle. Then, separation can be carried out in a magnetic field, for example, by "holding" the magnetic particles while the reaction fluid is removed therefrom. U.S. Pat. No. 3,933,997 to Hersh et al. discloses the use of an immunoadsorbent of anti-digoxin antibodies coupled through an intermediate silane to iron oxide particles. Nye et al., Clin. Chim. Acta. 69:387 (1976), describe antibodies covalently linked to polymer-coated iron oxide. Guesdon et al., Immunochem. 14:443 (1977), describe the use of magnetic polyacrylamideagarose-magnetite beads for use in solid-phase enzymeimmunoassay. Ithakissios et al., Clin. Chim. Acta. 84:69 (1978) and Clin. Chem. 23/11,2072 (1979) describe magnetic microparticles of a protein matrix containing magnetic material and use thereof in immunoassays.
U.S. Pat. Nos. 3,970,518 and 4,018,886, both to Giaever, disclose the presence of a monomolecular coating of antibody and protein, respectively, on magnetic particles ranging from colloidal size to about 10 microns to detect biological particles which will specifically interact with the coated material. The Giaever patents contemplate the types of magnetic particles as follows:
Ferromagnetic, ferrimagnetic and superparamagnetic materials are useful in the practice of this invention. Other suitable magnetic materials include oxides, such as, for example, ferrites, perovskites, chromites and magnetoplumbites.
The single example in Giaever U.S. Pat. No. 4,018,886 uses nickel particles about 1 micron in diameter coated with bovine serum albumin. One disadvantage of using such relatively large particles of the magnetic material is that the particles will tend to adhere to one another after removal of the magnetic field because of residual magnetism. Also, pure magnetic material usually has a high density.
U.S. Pat. No. 3,985,649 to Eddelman describes ferromagnetic particles of (1) a ferromagnetic core coated with a biomaterial support such as glass, or porous glass, (2) ferromagnetic particles adhesively attached to the biomaterial support or (3) blending a very finely divided ferromagnetic substance with a support material such as a polymer, and then forming the ferromagnetic particles. A biologically active material can be affixed to the ferromagnetic particles for use in RIA techniques.
Those skilled in the art will recognize that the force on a suspended magnetic particle subjected to a magnetic field is directed to move the particle to stronger field regions (typically towards the pole of a magnet) and that the strength of the force depends both on the field gradient and magnetism induced in the particle by the field. Thus, for rapid separation, a strong separator and a highly magnetizable particle appear preferable.